Cancer is a term used to describe a group of malignancies that all share the common trait of developing when cells in a part of the body begin to grow out of control. Most cancers form as tumors, but can also manifest in the blood and circulate through other tissues where they grow. Cancer malignancies are most commonly treated with a combination of surgery, chemotherapy, and/or radiation therapy. The type of treatment used to treat a specific cancer depends upon several factors including the type of cancer malignancy and the stage during which it was diagnosed.
Busulfan is a common cytotoxic agent that is used for the treatment of chronic myelogenous leukemia and high-dose pre-transplant conditioning. This chemotherapeutic agent has the formula:
This compound has been associated with debilitating side effects such as mucositis, hepatic veno-occlusive disease and myelosuppression. By monitoring the levels of busulfan in the body and adjusting the dose, these side effects can be better controlled and limited in patients.
Routine therapeutic drug management of busulfan requires the use of simple automated tests such as immunoassays which are adaptable to general laboratory equipment. In general these immunoassay are performed on fluid samples taken from a patient who is being treated with the busulfan so as to monitor his therapy. In so doing the health facilities, such as hospitals, laboratories and doctors offices use immunoassays by means of kits for performing such immunoassays. In order to carry out these assays, kits containing reagents, standards and instructions for carrying out the immunoassay are provided by their manufacturer. In this way the immunoassay can be performed at these health facilities. These kits usually contain the necessary reagents in separate containers for carrying out the busulfan immunoassay on the patient samples.
In order to quantify the amount of busulfan in a patient sample, a set of standards, each standard containing a known amount or concentration of busulfan, are placed in the assay kit or in a separate calibration kit to correlate the results of the immunoassay on the patient sample with the results achieved through subjecting the standards containing known amounts or known concentrations of busulfan to the same immunoassay to which the sample was subjected. In these kits the set of standards contains a sufficient number of standards of different concentration of busulfan, so that when all of the standards are subjected to the immunoassay, the results achieved with respect to the all of the standards can be plotted against the different concentrations to provide a curve or a straight line.
In this manner by means of this immunoassay, the results with respect to the patient's sample can be compared and correlated through this plot to the results achieved by means of these standards with known concentrations of the busulfan. Locating on this plot, where the results obtained with the patent sample fall, will determine the amount of busulfan in the patient samples.
A major flaw in these kits or in the calibration kits for determining busulfan in a patient's sample is that the busulfan in the standard is unstable and after a prolonged period of time decomposes. Busulfan is a bifunctional alkylating agent that has two labile methanesulfonate groups that are attached at the opposite ends of a saturated four carbon chain. These two groups are readily hydrolyzed in aqueous media especially in the presence of nucleophiles. Studies have shown that busulfan decomposes significantly at 37° C. with a half life of 14 hours. At room temperature for 5 days, more than 60% of busulfan is decomposed.
In developing calibrators or controls of busulfan for use as standards in a quantitative assay, special care has to be taken so that the busulfan does not decompose and give inaccurate results. In practice calibrator matrix material is spiked at various levels with busulfan and the material is then aliquoted into smaller vials and frozen at −20° C. When an assay needs to be run, one of the aliquots is thawed and used in the assay that day. It cannot be keep at room temperature for more than a few hours and even at 4° C. the calibrator decomposes within a day. This adds a complexity and expense to laboratories in addition to causing doubt on the results due to the decomposition of the material used as a calibrator. The instability of busulfan calibrators and controls can often lead to erroneous results. Therefore having a stabilized material for use as a standard would allow for a great ease of use and more confidence in the results.